Serratia marcescens nuclease

{{Infobox enzyme

| Name = Serratia marcescens nuclease

| EC_number = 3.1.30.2

| CAS_number = 9025-65-4

| GO_code =

| image =

| width =

| caption =

}}

{{Infobox nonhuman protein|Organism=Serratia marcescens|Symbol=nucA|UniProt=P13717}}

Serratia marcescens nuclease ({{EnzExplorer|3.1.30.2}}, endonuclease (Serratia marcescens), barley nuclease, plant nuclease I, nucleate endonuclease) is an enzyme.{{cite journal | vauthors = Mikulski AJ, Laskowski M | title = Mung bean nuclease I. 3. Purification procedure and (3') omega monophosphatase activity | journal = The Journal of Biological Chemistry | volume = 245 | issue = 19 | pages = 5026–5031 | date = October 1970 | doi = 10.1016/S0021-9258(18)62813-3 | pmid = 4319109 | doi-access = free }}{{cite journal | vauthors = Stevens A, Hilmoe RJ | title = Studies on a nuclease from Azotobacter agilis. I. Isolation and mode of action |journal = Journal of Biological Chemistry |date = 1960 |volume = 235 | issue = 10 |pages = 3016–3022 | doi = 10.1016/S0021-9258(18)64581-8 | doi-access = free }}{{cite journal | title = Studies on a nuclease from Azotobacter agilis. II. Hydrolysis of ribonucleic and deoxyribonucleic acids | vauthors = Stevens A, Hilmoe RJ |journal = Journal of Biological Chemistry |date = 1960 |volume = 235 | issue = 10 |pages = 3023–3027 | doi = 10.1016/S0021-9258(18)64582-X | doi-access = free }}{{cite journal | vauthors = Wechter WJ, Mikulski AJ, Laskowski M | title = Gradation of specificity with regard to sugar among nucleases | journal = Biochemical and Biophysical Research Communications | volume = 30 | issue = 3 | pages = 318–322 | date = February 1968 | pmid = 4296679 | doi = 10.1016/0006-291x(68)90453-1 }} This enzyme catalyses the following chemical reaction

: Endonucleolytic cleavage to 5'-phosphomononucleotide and 5'-phosphooligonucleotide end-products

Hydrolyses double- or single-stranded substrate DNA or RNA. It is a representative of the DNA/RNA non-specific endonuclease family.

It is commercially available.

Characteristics

Serratia nuclease was first purified from its native source in 1969.{{cite journal | vauthors = Nestle M, Roberts WK | title = An extracellular nuclease from Serratia marcescens. I. Purification and some properties of the enzyme | journal = The Journal of Biological Chemistry | volume = 244 | issue = 19 | pages = 5213–5218 | date = October 1969 | pmid = 4899013 | doi = 10.1016/s0021-9258(18)63648-8 | publisher = Elsevier BV | doi-access = free }} It was cloned in 1987 and shown to consist of a 266 protein precursor,{{cite journal | vauthors = Ball TK, Saurugger PN, Benedik MJ | title = The extracellular nuclease gene of Serratia marcescens and its secretion from Escherichia coli | journal = Gene | volume = 57 | issue = 2–3 | pages = 183–192 | year = 1987 | pmid = 3319779 | doi = 10.1016/0378-1119(87)90121-1 | publisher = Elsevier BV }} which is further cleaved and secreted as a 245 amino acid active nuclease.{{cite journal | vauthors = Biedermann K, Jepsen PK, Riise E, Svendsen I | title = Purification and characterization of a Serratia marcescens nuclease produced by Escherichia coli | journal = Carlsberg Research Communications | volume = 54 | issue = 1 | pages = 17–27 | year = 1989 | pmid = 2665765 | doi = 10.1007/bf02910469 | publisher = Springer Science and Business Media LLC | s2cid = 12831178 | doi-access = free }} Its active form in solution is a homodimer.{{cite journal | vauthors = Benedik MJ, Strych U | title = Serratia marcescens and its extracellular nuclease | journal = FEMS Microbiology Letters | volume = 165 | issue = 1 | pages = 1–13 | date = August 1998 | pmid = 9711834 | doi = 10.1111/j.1574-6968.1998.tb13120.x | publisher = Oxford University Press (OUP) | doi-access = free }} It has two disulfide bonds, the first between cysteine 30 & 34 and the second between cysteine 222 & 264. Reduction of these disulfides or site directed mutagenesis of their residues to serine, specifically the first one, leads to a large loss in nuclease activity, and a loss of the ability to reversibly regain activity after inactivating 40-60˚C heat treatments. It has a much higher catalytic efficiency than other nucleases, about 4 times greater than staphylococcal nuclease, and about 34 times greater than bovine pancreatic DNase I. The enzyme cleaves single or double stranded DNA and RNA with similar rates, so long as the substrate DNA or RNA contains no fewer than 5 nucleotides (or basepairs). Magnesium (II) (Mg²⁺) is an essential cofactor for its nuclease activity. Serratia nuclease is activated by up to 4M urea.{{cite web |title=Benzonase® Nuclease - Effective removal of nucleic acids and viscosity reduction from protein solutions |url= https://www.sigmaaldrich.com/deepweb/assets/sigmaaldrich/product/documents/342/154/benzonase-nuclease-ms.pdf | work = EMD Biosciences | publisher = SigmaAldrich |access-date=29 April 2023}} At 5M urea the initial activity is decreased from its peak although still above its baseline, and the enzyme is significantly inhibited after 60 minutes. At 6M urea, the nuclease activity is below baseline and almost completely inactivated within 60 minutes. At 7M the nuclease becomes essentially completely inactivated within 15 minutes, but significant and workable degradation of nucleic acids can occur before the nuclease is inactivated. 8M urea causes a complete inactivation of the enzyme within 5 minutes.

=Optimal conditions<ref name="EMD_datasheet" />=

Condition	Optimal¹	Effective²
class="wikitable"
Mg²⁺ concentration	1 - 2 mM	1 - 10 mM
pH	8.2 - 9.2	6.0 - 10.0
Temperature	37˚C	0 - 42˚C
Dithiothreitol (DTT)	< 100 mM	> 100 mM
β-Mercaptoethanol (BME)	< 100 mM	> 100 mM
Monovalent cation concentration (Na⁺, K⁺, etc.)	0 - 20 mM	0 - 150 mM
PO₄^3-	0 - 10 mM	0 - 100 mM
Urea	< 4M	> 4M

1="Optimal" is the condition in which Serratia nuclease retains >90 % of its activity.

2="Effective" is the condition in which Serratia nuclease retains >15 % of its activity.

=Inhibitory conditions=

Some inhibitory conditions are known:

>300 mM monovalent cations (Na⁺, K⁺, etc.)
>100 mM phosphate
>100 mM ammonium sulfate
>100 mM guanidine HCl
>2 mM EDTA
>4 mM EGTA
>0.4% w/v Triton X-100 (no effect below 0.4%, slight activation above 0.4%)
>0.4% w/v Sodium deoxycholate (70% activity at 0.4%, steady inactivation below and above 0.4%)
>0.1% w/v SDS (inactivation kinetics allow for Serratia nuclease to still degrade some nucleic acids before inactivation)

Use in biotechnology

Given its high activity, high stability & reversible inactivation to heat treatments, rate enhancement or otherwise compatibility with some denaturing reagents like urea, Serratia nuclease was recognized early on to have industrial & commercialization potential. A patent covering the recombinant expression of Serratia nuclease in E. coli was submitted by Benzon Pharma in 1986, granted in 1992, & expired in 2006.{{cite patent | country = EP | number = 0229866A1 | title = Bacterial enzymes and method for their production | gdate = 9 December 1992 | inventor = Molin S, Givskov M, Riise E | assign1 = Benzon Pharma AS | assign2 = Takeda Pharma AS | url=https://patents.google.com/patent/EP0229866A1/en?oq=EP0229866 }} This recombinant Serratia nuclease was commercialized as Benzonase, and is still available from and a registered trademark of Merck KGaA.{{cite web | title=Benzonase® Nuclease HC, Purity > 99% - 71206 | website=MilliporeSigma | url=https://www.emdmillipore.com/US/en/product/Benzonase-Nuclease-HC-Purity-990-0,EMD_BIO-71206 | access-date=2023-04-29}} Notably, the patented sequence{{cite web | title=UniProt | website=UniProt | url=https://www.uniprot.org/uniprotkb/A0A8G2LD96/entry | access-date=2023-04-29}} for Benzonase is slightly different (1 amino acid substitution) from the Serratia marcescens nuclease which was cloned publicly.{{cite web | title=UniProt | website=UniProt | url=https://www.uniprot.org/uniprotkb/P13717/entry | access-date=2023-04-29}}

As the benzonase patent is now expired, and in fact was never submitted nor granted in the United States, several commercial alternatives for recombinantly produced Serratia marcescens nuclease are now available:

Basemuncher, from Westburg Life Sciences{{cite web | title=Basemuncher Benzonase | website=Westburg | url=https://www.westburg.eu/products/protein-analysis/protein-expression-purification/extraction-and-lysis-reagents/basemuncher-benzonase | access-date=2023-04-29}}
Benzo Nuclease, from Tinzyme{{cite web | title=Benzo Nuclease | website=Tinzyme Ltd – Enzymes, dNTP and rNTP | date=2021-12-24 | url=https://tinzyme.com/benzo-nuclease/benzo-nuclease/ | access-date=2023-04-29}}
Benz-Neburase, from GenScript{{cite web | title=Benz-Neburase™, His | website=GenScript | date=2021-08-12 | url=https://www.genscript.com/enzyme/Z03626-Benzone_Nuclease.html | access-date=2023-04-29}}
Decontaminase, from AG Scientific{{cite web | title=B-1400-5KU - Decontaminase™, 5 KU | website=AG Scientific | date=2022-12-13 | url=https://agscientific.com/products/enzymes/d-l-enzymes/decontaminase%E2%84%A2-5-ku.html | access-date=2023-04-29}}
Denarase, from c-LEcta{{cite web | title=Denarase | website=c-LEcta | date=2022-12-13 | url=https://denarase.c-lecta.com | access-date=2023-04-29}}
Dr. Nuclease, from Syd Labs{{cite web | title=Benzonase Nuclease Alternative, DENARASE Nuclease Alternative | website=Syd Labs | date=2020-05-01 | url=https://www.sydlabs.com/recombinant-dr-nuclease-p15425.htm | access-date=2023-04-29}}
GENIUS Nuclease, from ACROBiosystems{{cite web | title=GENIUS™Nuclease DMF Filed | website=ACROBiosystems | url=https://www.acrobiosystems.com/P266-GENIUS™Nuclease-DMF-Filed.html | access-date=2023-04-29}}
Pierce Universal Nuclease, from Thermo Fisher Scientific{{cite web | title=Pierce™ Universal Nuclease for Cell Lysis | website=Thermo Fisher Scientific | date=2023-04-29 | url=https://www.thermofisher.com/order/catalog/product/88700 | access-date=2023-04-29}}
TurboNuclease, from Accelagen{{cite web | title=TurboNuclease | website=Accelagen | date=2023-04-29 | url=https://accelagen.com/TurboNuclease.htm | access-date=2023-04-29}}
MaxNuclease™, from KACTUS{{cite web | title=MaxNuclease™ Benzonase Nuclease Alterantive | website=KACTUS | date=2025-02-19 | url=https://kactusbio.com/pages/maxnuclease | access-date=2025-02-19}}

(A current notable non-producer is New England Biolabs){{cite web |last1=Biolabs |first1=New England |title=DNA Modifying Enzymes & Cloning Technologies - Exonucleases and Non-specific Endonucleases |url=https://www.neb.com/products/dna-modifying-enzymes-and-cloning-technologies#:~:text=Exonucleases |publisher=New England Biolabs |access-date=30 April 2023}}

References

External links

{{MeshName|Serratia+marcescens+nuclease}}
Serratia marcescens endonuclease at [http://www.protean.cz/en/recombinant-proteins/enzymes Protean Ltd.], highly active

Category:EC 3.1.30

Category:Nucleases